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At least 775 records · Page 43Linked to original sources
PubMed2005

Absence of non-specific effects of RNA interference triggered by long double-stranded RNA in mouse oocytes.

RNA interference (RNAi) is a conserved eukaryotic mechanism by which double-stranded RNA (dsRNA) triggers the sequence-specific degradation of homologous mRNAs. Recent concerns have arisen in mammalian systems about off-target effects of RNAi, as well as an interferon response. Most mammalian cells respond to long dsRNAs by inducing an antiviral response mediated by interferon that leads to general inhibition of protein synthesis and nonspecific degradation of mRNAs. Moreover, recent reports demonstrate that under certain conditions, short interfering RNAs (siRNAs, 21-25 bp) may activate the interferon system. Mouse oocytes and preimplantation embryos apparently lack this response, as potent and specific inhibition of gene expression triggered by long dsRNA is observed in these cells. In the present study, we analyzed the global pattern of gene expression by microarray analysis in transgenic mouse oocytes expressing long dsRNA and find no evidence of off-targeting. We also report that genes involved in the interferon response pathway are not expressed in mouse oocytes, even after exposure for an extended period of time to long dsRNA.

Animals↗
PubMed2004

Role of protein kinase R in double-stranded RNA-induced expression of nitric oxide synthase in human astroglia.

Environmental factor(s), such as viral infection, has been implicated as one of the triggering events leading to neuroinflammation in multiple sclerosis. This study underlines the importance of double-stranded RNA (dsRNA), the active component of a viral infection, in inducing the expression of inducible nitric oxide synthase (iNOS) in human astroglia. DsRNA in the form of synthetic polyinosinic-polycytidylic acid (poly IC) induced expression of iNOS and iNOS promoter-driven luciferase activity through activation of nuclear factor (NF)-kappaB and CCAAT/enhancer-binding proteinbeta (C/EBPbeta). In addition, we show that inhibitors of protein kinase R attenuated iNOS by suppressing the activation of NF-kappaB but not C/EBPbeta. In contrast, knock down of p38 mitogen-activated protein kinase (MAPK) attenuated iNOS by suppressing the activation of C/EBPbeta but not NF-kappaB. This study delineates a novel role of dsRNA in inducing the expression of iNOS through dsRNA-activated protein kinase (PKR)-mediated activation of NF-kappaB and p38-mediated activation of C/EBPbeta in human astroglia that may participate in virus-induced neurological abnormalities.

Astrocytes↗
PubMed2003

Sequence dependence of substrate recognition and cleavage by yeast RNase III.

Yeast Rnt1p is a member of the double-stranded RNA (dsRNA) specific RNase III family of endoribonucleases involved in RNA processing and RNA interference (RNAi). Unlike other RNase III enzymes, which recognize a variety of RNA duplexes, Rnt1p cleaves specifically RNA stems capped with the conserved AGNN tetraloop. This unusual substrate specificity challenges the established dogma for substrate selection by RNase III and questions the dsRNA contribution to recognition by Rnt1p. Here we show that the dsRNA sequence adjacent to the tetraloop regulates Rnt1p cleavage by interfering with RNA binding. In context, sequences surrounding the cleavage site directly influence the cleavage efficiency. Introduction of sequences that stabilize the RNA helix enhanced binding while reducing the turnover rate indicating that, unlike the tetraloop, Rnt1p binding to the dsRNA helix may become rate-limiting. These results suggest that Rnt1p activity is strictly regulated by a combination of primary and tertiary structural elements allowing a substrate-specific binding and cleavage efficiency.

Base Sequence↗
PubMed2003

Rotavirus NSP2 interferes with the core lattice protein VP2 in initiation of minus-strand synthesis.

The rotavirus nonstructural protein NSP2 self-assembles into stable octameric structures that possess nonspecific affinity for single-stranded (ss)RNA and RNA-RNA helix-destabilizing and NTPase activities. Furthermore, NSP2 is a component of replication intermediates with replicase activity and plays a critical role in the packaging and replication of the segmented dsRNA genome of rotavirus. To better understand the function of the protein in genome replication, we examined the effect that purified recombinant NSP2 had on the synthesis of dsRNA by the open core replication system. The results showed that NSP2 inhibited the synthesis of dsRNA from viral mRNA in vitro, in a concentration-dependent manner. The inhibition was overcome by adding increasing amounts of viral mRNA or nonviral ssRNA to the system, indicating that the inhibition was mediated by the nonspecific RNA-binding activity of NSP2. Further analysis revealed that NSP2 interfered with the ability of the open core proteins, GTP, and viral mRNA to form the initiation complex for (-) strand synthesis. Additional experiments indicated that NSP2 did not perturb recognition of viral mRNA by the viral RNA polymerase VP1, but rather interfered with the function of VP2, a protein that is essential for (-) strand initiation and dsRNA synthesis and that forms the T = 1 lattice of the virion core. In contrast to initiation, NSP2 did not inhibit (-) strand elongation. Collectively, the findings provide evidence that the temporal order of interaction of RNA-binding proteins with viral mRNA is a crucial factor impacting the formation of replication intermediates.

Capsid Proteins↗
PubMed2003

Kunjin RNA replication and applications of Kunjin replicons.

The Kunjin virus (KUNV) has provided a useful laboratory model for Flavivirus RNA replication. The synthesis of progeny RNA(+) strands occurs via asymmetric and semiconservative replication on a template of recycling double-stranded RNA (dsRna) or replicative form (RF). Kinetics of viral RNA synthesis indicated a cycle period of about 15 min during which, on average, a single nascent RNA (+) strand displaces the pre-existing RNA(+) strand in the replicative intermediate. Data on the composition of the replication complex (RC) in KUNV-infected cells were obtained from several sources, including analyses of the partially-purified still active RC, immunogold labeling of cryosections using monospecific antibodies to the nonstructural proteins and to the dsRNA, radioimmunoprecipitations of cell lysates using antibodies to dsRNA and to an RC-associated cell marker, and pull-down assays of cell lysates using fusion proteins GST-NS2A and GST-NS4A. These results yeilded a consensus composition of NS1, NS2A, NS3, NS4A, and NS5 strongly associated with the dsRNA template. The RC was located in induced membranes described as vesicle packets. The RNA-dependent RNA polymerase activity late in infection did not require continuing protein synthesis. Replication of genomic RNA was completely dependent on the presence of conserved complementary or cyclization sequences near the 5' and 3' ends. Assembly of the RC during translation in cis and the relationships, particularly those of NS1 and NS5 among the components, were deduced from an extensive set of complementation experiments in trans involving mutations/deletions in all the nonstructural proteins and use of KUN or alphahavirus replicons as helpers. The KUN replicon has found useful applications also as a noncytopathic vector for the continuing expression of foreign genes, delivered either as packaged RNA or as plasmid DNA.

Amino Acid Sequence↗
PubMed2007

Coupling of rotavirus genome replication and capsid assembly.

The Reoviridae family represents a diverse collection of viruses with segmented double-stranded (ds)RNA genomes, including some that are significant causes of disease in humans, livestock, and plants. The genome segments of these viruses are never detected free in the infected cell but are transcribed and replicated within viral cores by RNA-dependent RNA polymerase (RdRP). Insight into the replication mechanism has been provided from studies on Rotavirus, a member of the Reoviridae whose RdRP can specifically recognize viral plus (+) strand RNAs and catalyze their replication to dsRNAs in vitro. These analyses have revealed that although the rotavirus RdRP can interact with recognition signals in (+) strand RNAs in the absence of other proteins, the conversion of this complex to one that can support initiation of dsRNA synthesis requires the presence and partial assembly of the core capsid protein. By this mechanism, the viral polymerase can carry out dsRNA synthesis only when capsid protein is available to package its newly made product. By preventing the accumulation of naked dsRNA within the cell, the virus avoids triggering dsRNA-dependent interferon signaling pathways that can induce expression and activation of antiviral host proteins.

Amino Acid Sequence↗
PubMed2005

RNA interference spreading in C. elegans.

The phenomenon of RNA interference (RNAi) occurs in eukaryotic organisms from across the boundaries of taxonomic kingdoms. In all cases, the basic mechanism of RNAi appears to be conserved--an initial trigger [double-stranded RNA (dsRNA) containing perfect homology over at least 19-21/bp with an endogenous gene] is processed into short interfering RNA (siRNA) molecules and these siRNAs stimulate degradation of the homologous mRNA. In the vast majority of species, RNAi can only be initiated following the deliberate introduction of dsRNA into a cell by microinjection, electroporation, or transfection. However, in the nematode worm Caenorhabditis elegans, RNAi can be simply initiated by supplying dsRNA in the surrounding medium or in the diet. Following uptake, this dsRNA triggers a systemic effect, initiating RNAi against the corresponding target gene in tissues that are not in direct contact with the external milieu. This phenomenon of systemic RNAi, or RNAi spreading, is notably absent from mammalian species, a fact that is likely to prove a substantial barrier to the wider use of RNAi as a clinical therapy. An understanding of the mechanism of systemic RNAi is therefore of considerable importance, and several advances of the last few years have begun to shed light on this process. Here we review our current understanding of systemic RNAi in C. elegans and draw comparisons with systemic RNAi pathways in other organisms.

Animals↗
PubMed2003

Efficiency and specificity of RNA interference generated by intra- and intermolecular double stranded RNA in Trypanosoma brucei.

In many eukaryotes, double-stranded (ds) RNA leads to specific degradation of RNA of cognate sequence, a process termed RNA interference (RNAi). Here we used the protozoan Trypanosoma brucei as a model to investigate efficiency and specificity of RNAi generated by expression of long dsRNA of PFRA and PFRC genes, which code for flagellar proteins required for cell motility. Consequences of RNAi were monitored at all three levels: target RNA expression, protein expression and phenotype observation, using population or individual cell analysis. Expression of PFRA dsRNA from an inverted repeat was extremely efficient, knocking down PFRA RNA and PFRA protein, and producing a severe paralysis phenotype. Silencing by expression of PFRA dsRNA using a dual facing promoter system was also very efficient, producing a clear phenotype, although low amounts of PFRA RNA and PFRA protein were detected. Expression via the dual facing promoters of PAR2 dsRNA (83% overall identity with PFRA, including nine blocks of >20 nt total identity) did not produce significant reduction of total amounts of PFRA RNA or PFRA protein. However, individual cell analysis by immunofluorescence revealed that 10-60% cells (depending on subclones) exhibited lower PFRA amounts in their flagellum, producing a reduced-motility phenotype.

Animals↗
PubMed2001

The H1 double-stranded RNA genome of Ustilago maydis virus-H1 encodes a polyprotein that contains structural motifs for capsid polypeptide, papain-like protease, and RNA-dependent RNA polymerase.

The Ustilago maydis viral (UmV) genome consists of three distinct size groups of double-stranded RNA (dsRNA) segments: H (heavy), M (medium), and L (light). The H segments have been suggested to encode all essential viral proteins, but without any molecular evidences. As a preliminary step to understand viral genomic organization and the molecular mechanism governing gene expression in UmV, we determined the complete nucleotide sequence of the H1 dsRNA genome in P1 viral killer subtype. The H1 dsRNA genome (designated UmV-H1) contained a single open reading frame that encodes a polyprotein of 1820 residues, which is predicted to be autocatalytically processed by a viral papain-like protease to generate viral proteins. The amino-terminal region is the capsid polypeptide with a predicted molecular mass of 79.9 kDa. The carboxy-terminal region is the RNA-dependent RNA polymerase (RDRP) that has a high sequence homology to those of the totiviruses. The H2 dsRNA also encodes a distinct RDRP, suggesting that UmV is a complex virus system like the Saccharomyces cerevisiae viruses ScV-L1 and -La.

Amino Acid Sequence↗
PubMed2001

Short 5'-phosphorylated double-stranded RNAs induce RNA interference in Drosophila.

Double-stranded (ds) RNA causes the specific degradation of homologous RNAs in a process called "RNA interference (RNAi)"[1-4]; this process is called "posttranscriptional gene silencing (PTGS)" in plants [5-7]. Both classes of gene silencing have been reviewed extensively [8-13]. The duplex RNA becomes processed by Dicer [14] or another RNase III-like enzyme to short dsRNA fragments of about 21-23 nucleotides (nt) [15], which are incorporated in the RNA-induced silencing complex (RISC)[16] that directs target-specific RNA degradation [17, 18]. Here, we show that different synthetic dsRNA cassettes, consisting of two 5'-phosphorylated RNA strands of 22 nt each, can initiate RNAi in Drosophila embryos. The cassettes were active at similar quantities required to initiate RNAi by conventional dsRNA. Their sequence specificity was confirmed using synthetic dsRNA cassettes for two different genes, Notch and hedgehog; each time, only the relevant embryonic phenotype was observed. Introduction of point mutations had only a moderate effect on the silencing potential, indicating that the silencing machinery does not require perfect sequence identity. 5'-phosphorylated synthetic RNA was more active than its hydroxylated form. Substitution of either RNA strand by DNA strongly reduced activity. Synthetic cassettes of siRNA will provide a new tool to induce mutant phenotypes of genes with unknown function.

5' Untranslated Regions↗
PubMed2001

Crystallographic and modeling studies of RNase III suggest a mechanism for double-stranded RNA cleavage.

BACKGROUND: Aquifex aeolicus Ribonuclease III (Aa-RNase III) belongs to the family of Mg(2+)-dependent endonucleases that show specificity for double-stranded RNA (dsRNA). RNase III is conserved in all known bacteria and eukaryotes and has 1-2 copies of a 9-residue consensus sequence, known as the RNase III signature motif. The bacterial RNase III proteins are the simplest, consisting of two domains: an N-terminal endonuclease domain, followed by a double-stranded RNA binding domain (dsRBD). The three-dimensional structure of the dsRBD in Escherichia coli RNase III has been elucidated; no structural information is available for the endonuclease domain of any RNase III. RESULTS: We present the crystal structures of the Aa-RNase III endonuclease domain in its ligand-free form and in complex with Mn(2+). The structures reveal a novel protein fold and suggest a mechanism for dsRNA cleavage. On the basis of structural, genetic, and biological data, we have constructed a hypothetical model of Aa-RNase III in complex with dsRNA and Mg(2+) ion, which provides the first glimpse of RNase III in action. CONCLUSIONS: The functional Aa-RNase III dimer is formed via mainly hydrophobic interactions, including a "ball-and-socket" junction that ensures accurate alignment of the two monomers. The fold of the polypeptide chain and its dimerization create a valley with two compound active centers at each end of the valley. The valley can accommodate a dsRNA substrate. Mn(2+) binding has significant impact on crystal packing, intermolecular interactions, thermal stability, and the formation of two RNA-cutting sites within each compound active center.

Amino Acid Sequence↗
PubMed2002

Transmission of double-stranded RNA in Heterobasidion annosum.

Transmission of dsRNA viruses between homo- and heterokaryotic mycelia paired on agar plates and into conidia has been studied in Heterobasidion annosum. Horizontal transmission of dsRNA occurred between both homo- and heterokaryotic isolates, as well as between isolates belonging to different intersterility groups. The proportions of vertical transmission into conidia were 3% and 55%, respectively, for the two isolates included in the study. RT-PCR of dsRNA and PCR-RFLP of mitochondrial markers were used to confirm transmission of dsRNA between the cytoplasms of different mycelia. The identity of nuclei and nuclear migration during experiments were verified using PCR-RFLP of several nuclear markers.

Basidiomycota↗
PubMed2003

RNAse III-mediated degradation of unspliced pre-mRNAs and lariat introns.

Double-stranded RNA (dsRNA) has emerged as a modulator of gene expression, from gene silencing to antiviral responses. Here we show that dsRNA stem-loop structures found in intronic regions of the S. cerevisiae RPS22B and RPL18A transcripts trigger degradation of unspliced pre-mRNAs and lariat introns and can control the level of mRNA produced from these intron-containing genes. The dsRNA regions are cleaved by Rnt1p, the yeast ortholog of RNase III, which creates an entry site for complete degradation by the Xrn1p and Rat1p exonucleases and by the nuclear exosome. These results identify an alternative discard pathway for precursors and products of the splicing machinery and a physiological function for dsRNA in eukaryotic RNA catabolism.

Base Sequence↗
PubMed2001

RNA interference: mechanisms and applications.

RNA interference (RNAi) is a phenomenon induced by double-stranded RNA (dsRNA) in which gene expression is inhibited through specific degradation of mRNA. The mechanism involves conversion of dsRNA into short RNAs that direct ribonucleases to homologous mRNA targets. This process is related to normal defence against viruses and mobilisation of transposons. Treatment with dsRNA has become an important method for analysing gene functions in invertebrate organisms. RNAi has also been demonstrated in several vertebrate species but with lower efficiency. Development of procedures for in vivo production of dsRNA may provide efficient tools for tissue- and stage-specific gene targeting.

Animals↗
PubMed1993

Mutational analysis of a ribonuclease III processing signal.

A mutational approach was employed to identify sequence and structural elements in a ribonuclease III processing signal that are important for in vitro enzymatic cleavage reactivity and selectivity. The substrate analyzed was the bacteriophage T7 R1.1 processing signal, a 60 nucleotide irregular RNA hairpin exhibiting an upper and lower dsRNA stem, separated by an asymmetric internal loop which contains the scissile phosphodiester bond. Altering the length of either the upper or lower dsRNA segment in R1.1 RNA dose not change the site of RNase III cleavage. However, decreasing the size of either the upper or lower dsRNA segment causes a progressive inhibition of processing reactivity. Omitting monovalent salt from the reaction buffer promotes cleavage of otherwise unreactive R1.1 deletion mutants. Accurate processing is maintained with R1.1 variants containing specific point mutations, designed to disrupt Watson-Crick (WC) base-pairing in a conserved sequence element within the upper dsRNA stem. The internal loop is not required for processing reactivity, as RNase III can accurately and efficiently cleave R1.1 variants in which this structure is WC base-paired. Moreover, an additional cleavage site is utilized in these variants, which occurs opposite the canonical site, and is offset by two nucleotides. The fully base-paired R1.1 variants form a stable complex with RNase III in Mg(2+)-free buffer, which can be detected by a gel electrophoretic mobility shift assay. In contrast, the complex of wild-type R1.1 RNA with RNase III is unstable during nondenaturing gel electrophoresis. Thus, a functional role of the T7 R1.1 internal loop is to enforce single enzymatic cleavage, which occurs at the expense of RNase III binding affinity.

Bacteriophage T7↗
PubMed1991

Electrophoretic and hydrodynamic properties of duplex ribonucleic acid molecules transcribed in vitro: evidence that A-tracts do not generate curvature in RNA.

We have developed a T7 RNA polymerase based transcription system for the production of fully complementary RNA molecules (i.e., molecules capable of forming blunt-ended duplex species) as the direct products of transcription, thus rendering unnecessary the enzymatic removal of single-stranded ends. A combined gel electrophoretic and hydrodynamic analysis of a 180 bp double-stranded (ds) RNA molecule containing four A5-tracts in approximate phase coherence with the helix repeat provides no indication that the helix axis is curved, in sharp contrast to DNA molecules containing phased A-tracts. The electrophoretic behavior of dsRNA molecules reveals that their mobilities in nondenaturing acrylamide gels are approximately 10-20% lower than the corresponding mobilities of duplex DNA, in accord with earlier observations in the literature. Furthermore, the relative mobilities are only slightly modulated by gel concentration, the concentration of monovalent salt, or the presence of spermidine and/or Mg2+. The reduced mobilities are not caused by increased contour length, since direct hydrodynamic measurements using transient electric birefringence indicate that the average helix rise, h, of the dsRNA molecules examined in the current study is 2.8 +/- 0.1 A/bp. The reduced electrophoretic mobilities, extrapolated to zero acrylamide concentration, are consistent with the lower residual charge predicted for dsRNA by counterion condensation theory. Finally, birefringence measurements indicate that dsRNA is only marginally stiffer than DNA, with a persistence length of ca. 500-700 A.

Base Sequence↗
PubMed1984

Isolation of a heme-controlled inhibitor of translation that blocks the interaction between messenger rna and eukaryotic initiation factor 2.

A heme-controlled inhibitor of translation was isolated from the S-100 of rabbit reticulocytes by a novel procedure including chromatography on double-stranded ribonucleic acid (dsRNA)-cellulose. The inhibitor thus purified is extremely active and functionally resembles previously studied heme-controlled inhibitor preparations in terms of kinetics and extent of inhibition of translation, relief of inhibition by eukaryotic initiation factor 2 (eIF-2), relief of inhibition by 2-aminopurine, and preferential inhibition of alpha-over beta-globin synthesis. The action of this inhibitor on translation is resistant to treatment with bacterial alkaline phosphatase, micrococcal nuclease, or trypsin and to incubation at 95 degrees C, pH 2 or pH 12. The inhibitor not only is retained on DEAE-cellulose, phosphocellulose, and dsRNA-cellulose but also exhibits a high affinity for the dye Cibacron Blue, properties that suggest that it may be a protein. Unlike previously described heme-controlled inhibitor preparations, or preparations that did not pass over dsRNA-cellulose, the inhibitor recovered upon dsRNA-cellulose chromatography does not exhibit eIF-2 kinase activity. The inhibitor does not block ternary complex formation between eIF-2, methionyl-tRNAfMet, and GTP but inhibits the ability of eIF-2 to form a complex with labeled globin mRNA. In the presence of inhibitor, the formation of mRNA/eIF-2 complexes can be restored effectively by an excess of eIF-2 but not by an excess of mRNA. The inhibitor thus appears to block the interaction between eIF-2 and mRNA not by competing with eIF-2 for a binding site on mRNA but, instead, by acting on eIF-2 itself.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1981

Inhibition of double-stranded ribonucleic acid activated protein kinase and 2',5'-oligo(adenylic acid) polymerase by ethidium bromide.

The activation of two enzymes induced by interferon, a protein kinase and the 2',5'-oligo(adenylic acid) polymerase [2'5'-oligo(A) polymerase], is inhibited by ethidium bromide. The activation of these enzymes requires double-stranded RNA (dsRNA), and binding of ethidium to dsRNA inhibits the activation process. This was shown by determining the concentration of ethidium inhibitory for poly(A) . poly(U)- and poly(I) . poly(C)-activated reactions. Activation of both protein kinase and 2',5'-oligo(A) polymerase is inhibited by much lower concentrations of ethidium with the former polymer as activator than with the latter polymer. Correspondingly, in the presence of magnesium, ethidium binds with much greater affinity to poly(A) . poly(U) than to poly(I) . poly(C). Synthesis of 2',5'-oligo(A) with poly(A) . poly(U) as activator is arrested by adding low ethidium concentrations, but it is resumed upon addition of poly(I) . poly(C). Kinase activity, however, is not inhibited when ethidium is added after the activating dsRNA. This suggests that the kinase interacts with dsRNA in a manner different from the 2',5'-oligo(A) polymerase interaction.

2',5'-Oligoadenylate Synthetase↗